PROJECT SUMMARY/ABSTRACT Triple-negative breast cancer (TNBC) is a devastating disease accompanied with substantial morbidity and mortality with very few tools in the clinical armamentarium to improve outcomes. The PI3K pathway, which mediates cancer proliferation and survival, is known to be dysregulated in the majority of TNBCs. However, single agent inhibition of PI3K in TNBC patients has shown only a modest clinical benefit, contrary to the theory of oncogene addiction. Recently published data from our group demonstrates that when PI3K is inhibited by a small molecule, an immediate compensatory up-regulation of the Wnt pathway occurs. The Wnt pathway is well known known for its role in cancer metastases and can confer resistance to initial PI3K inhibition. Simultaneous dual targeting of both pathways results in significant synergistic efficacy against TNBC models in vitro and in vivo. Based on these data, we have initiated a Phase I clinical trial using Gedatolisib (PI3K/mTOR inhibitor) and PTK7-ADC (Wnt pathway) for patients with metastatic TNBC (NCT03243331). Using correlative tissue samples from our trial, the overall objective of this proposal is to confirm Wnt pathway compensation in patients treated with Gedatolisib, and determine preliminary biomarkers of response to the combination of Gedatolisib + PTK7-ADC. We hypothesize that Wnt pathway compensation will occur in both tumor tissues via analysis of matched pre- vs post-treatment biopsies as well as in circulating tumor cells (CTCs). We further hypothesize that the degree of induction and expression of PTK7 as well as genomic aberrations in the PI3K pathway will predict which patients will respond to the combination. The rationale for this proposal is to confirm our preclinical observations of compensatory mechanisms to PI3K inhibition in TNBC patients as well as provide guidance for marker-based patient selection for a larger randomized Phase II trial of the combination that is planned. We will test our hypotheses via the following specific aims:(1) To determine tumoral expression and compensatory induction of the Wnt pathway in metastatic patients treated with the PI3K/mTOR inhibitor, Gedatolisib; and (2) To determine preliminary biomarkers of response to Gedatolisib and PTK7-ADC. The expected outcome of this work is an enhanced understanding and confirmation of immediate compensatory mechanisms occurring in patients? tumors and matched CTCs in response to PI3K inhibition. In addition, we will receive preliminary insights into biomarkers of response that will guide our future trials. This contribution is significant because it is an important step in a continuum of work focused on ushering-in a new therapeutic strategy for metastatic TNBC.